MicroRNAs (miRNAs) are a group of newly discovered non-coding RNAs and can modulate transcription and stability of certain or some target mRNAs by binding to 3's non-coding regions (3's UTRs) of the complementary antisense mRNA thereof in a sequence-specific manner.
Most of miRNAs, such as let-7 RNA, miR-1, miR-34, miR-60, and miR-87, are highly conservative in invertebrates and vertebrates, implicating that they may recognize multiple sites and (or) multiple target sequences of genes with conservative function.
Another subclass of miRNA, small temporal RNA (stRNA, such as lin-4 and let-7), has been was identified based on the analysis on the genome of Caenorhabditis elegans. stRNA plays important role in the process of regulating development, such as neuron regeneration, Dauer larva formation, vulva formation, and the terminal differentiation of hypodermal cells.
miRNAs are typically formed by the excision of folded RNA precursors of 60-70 nucleotides. Some miRNAs can be detected upon expression, while some detected only at the peak of expression.
Generally, only one of the strands of hairpin structure was excised and accumulated, presumably because miRNA precursors could be protected by binding proteins thereof against degradation. It is conjectured that these binding proteins may modulate the translational suppression of miRNA. Mature miRNAs can not be formed from the miRNA precursors unless Dicer RNase III and enzymes of Argonaute family are involved in the reaction.
MicroRNAs play important roles in many biological events such as growth, fission, differentiation, development, apoptosis and diseases. Up to now, more than 1200 microRNAs have been discovered in human cells. These miRNAs are involved in regulating more than 60% genes in human.
miRNAs are important biomarkers for the classification of tumors, diagnosis, prediction of diseases and assessment of prognosis. Tumor-associated miRNAs in serum or plasma have been served as biomarkers for tumor diagnosis. The detection and quantification of miRNA are important tools for the discovery of some target genes and pathways, study on disease mechanism, assessment of drug safety and efficacy, disease diagnosis and prognosis. Thus, it is very important to determine the amount of each miRNA in specific cells.
In some cases, it is more important to quantify miRNA, for example, compare levels of miRNA in different tissues or compare the change of miRNA in tissue before and after application of an external cause (a chemical or physical treatment). Because miRNA family with similar sequence will be present in the same cell, it is important to have a detection method with high sensitivity and good specificity. Moreover, it may be beneficial to have methods suitable for high throughput screening samples, e.g. homogeneous methods, and multiplexed methods.
Nowadays, a number of miRNA quantitative analysis methods have been developed, including miRNA chip arrays, SYBR Green® I-based miRNA qRT-PCR assays (Raymond, C. K., Roberts, B. S., Garrett-Engele, P., Lim, L. P., and Johnson, J. M (2005) Simple, quantitative primer-extension PCR assay for direct monitoring of microRNAs and short-interfering RNAs. RNA 11), stem-loop-based TaqMan assays (Chen C, Ridzon D A, Broomer A J, Zhou Z, Lee D H, Nguyen J T, Barbisin M, Xu N L, Mahuvakar V R, Andersen M R, Lao K Q, Livak K J, Guegler K J. 2005. Real-time quantification of microRNAs by stem-loop RT-PCR. Nucleic Acids Res 33(20):e179), beads-based assays, high throughput sequencing and the like. However, each of aforementioned methods has defects. For example, hybridization-based assays (microarray or micro-beads) suffer from low sensitivity and specificity; SYBR-Green® I-based methods suffer from high background and poor specificity; the specificity of stem-loop TaqMan assays is acceptable, however, it is expensive, time-consuming and suffers from deviation, so that it is difficult to be applied in high throughput screen.
EvaGreen® is a double strand DNA-binding dye, exhibiting low PCR inhibition, good melt curve, high thermal and light stability, non-mutagenicity and non-cytotoxicity. EvaGreen® has been used in the determination of melt curve and fluorescent quantitative PCR (Mao F, Leung W Y, Xin X. 2007. Characterization of EvaGreen® and the implication of its physicochemical properties for qPCR applications. BMC Biotechnol. 9; 7:76).
EvaGreen®-based PCR reaction liquids can be obtained from many companies, such as BioRad, Qiagen, Agilent and so on, and many labs have reported the use of EvaGreen™-based reaction liquids prepared in-house, however, there is no report or suggestion that EvaGreen® can improve specificity of reaction.
Summing up, there is no cost effective miRNA detection method available with high sensitivity and specificity.